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[
Biochemistry,
2007]
High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-active disulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminal redox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of a selenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineering techniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mouse mitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR from Caenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly, Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemical modifications were performed in this study: insertion of alanine residues between the cysteine residues of the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the position of the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating to insertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. In contrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues were inserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminus and a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic position by changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activity as the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate of DmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cys and Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, the results support our previous model of Sec as the leaving group during reduction of the C-terminus during the catalytic cycle.
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Biochemistry,
2008]
Most high M r thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys 1-Cys 2) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys 2 position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group p K a as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower p K a of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH.
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Curr Biol,
2002]
Genes for tiny RNAs have been found to be plentiful in the genomes of worms, flies, humans and probably all animals. Some of these microRNAs have been conserved through evolution, and many are expressed only at specific times or places. How they act is just beginning to be understood, but their importance to biology is likely to be great.
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[
International C. elegans Meeting,
1997]
We have been continuing to work on protocols for fixation, staining and embedding and want to share our results with the nematode community. Representative micrographs will be shown and protocols will be available for anyone interested.
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Comput Biol Med,
1985]
A system that can simultaneously track about 25 animals with the position of each determined once a second is described. The system includes a 6809 microprocessor, OS-9 operating system and application programs written in assembly and BASIC09. The movements and changes in direction of the subjects can be determined and displayed in real time. The system has proven to be valuable in studying the chemotaxis of nematodes and should be applicable to the study of other animals that can be viewed
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[
International C. elegans Meeting,
1997]
C. elegans can be used as a tool in the discovery of new insecticides. Lead Detection - Wild type C. elegans can be used for screening of unknown compounds. The ideal is to use the target species on its normal host plant. However, that may not be amenable to screening. C. elegans has its advantages: - short life cycle and ease of culturing means the C. elegans can be grown to a large number in a short time using inexpensive culture medium. It can be stored frozen between use. - Our initial study shows that C. elegans has a reasonably selective activity to a spectrum of insecticide standards. Mode Of Action (i) - a panel of C. elegans mutants (each with known resistance or sensitivity) can be used to define the mode of action of unknown compounds. - If a mutant responds in a characteristic way to a compound, it is indicative of a relevant mode of action. - If all mutants do not provide any characteristic response to a compound, it is indicative of a novel mode of action. - Behavioural symptoms can be very useful. - It can be useful for predicting resistance and cross-resistance. - It can be useful for grouping unknown compounds. Mode Of Action (ii) - the unknown compound with a putative novel mode of action can be used as selective agent after EMS mutagenesis to generate mutants. - It can be useful towards understanding the mode of action by carrying out further work on the mutant. - The existing extensive information on ACeDB will facilitate characterisation of the mutant. - The mutant can be screened against a spectrum of putative relevant mode of action insecticide standards to check for cross resistance.
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Nat Genet,
1997]
Traditional reverse genetics on yeast, mice and other organisms uses homologous recombination with transgenic DNA to interrupt a target gene. Here we report that target-selected gene inactivation can be be achieved in Caenorhabditis elegans with the use of chemical mutagens. We use PCR to selectively visualize deletions in genes of interest; the method is sensitive enough to permit detection of a single mutant among more than 15,000 wild types. A permanent frozen mutant collection of more than a million mutagenized animals has been established, and deletion mutants of several G-protein genes were isolated from it. The approach is suitable to be scaled up for systematic inactivation of all 17,000 C. elegans genes. Because it requires no transgenesis or cell culturing, it may also be applicable to small organisms usually considered to be outside the realm of reverse genetics (for example, other nematodes and insects). Any sequenced gene in any organism that can be handled in very large numbers can possibly be targeted in this way.
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Environ Pollut,
2008]
Whether the multiple biological toxicities from nickel exposure could be transferred to progeny has not been clarified. In this report, we explored the Caenorhabditis elegans to analyze the multiple toxicities of nickel and their possibly transferable properties. The nickel toxicity caused multiple biological defects in a concentration-dependent manner. Moreover, most of these toxicities could be transferred and could be only partially rescued in progeny. Some specific phenotypes in progeny were also found to exhibit no obvious rescue phenotypes or to show even more severe defects than their parents. The defects caused by nickel exposure could be classified into four groups according to their transferring properties. That is, the defects caused by nickel exposure could be largely, or partially, or unable to be rescued, or became even more severe in progeny animals. Therefore, most of the nickel exposure-caused defects can be transferred from parents to their progeny to different degrees in C. elegans.
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[
International C. elegans Meeting,
1997]
We will describe a variety of interesting germline and somatic promoter activity patterns and make some suggestions as to how you might be able to engineer your gene to be expressed in any of all of these patterns.
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Experientia,
1969]
Caenorhabditis briggsae, a free living soil nematode, can be grown axenically in a chemically defined medium if this is supplemented with proteinaceous materials. Only a few such materials, derived from chick embryo, bacteria, and liver have been found to be effective supplements. Since yeast cultures on agar support nematodes in vitro it seemed probable that an effective supplement could also be made from extracts of yeast.